RU1124544C - Heptapeptide possessing properties of psychostimulator of prolonged action with immunotropic activity - Google Patents

Description

 The invention relates to a new biologically active compound - a heptapeptide having the properties of a long-acting psychostimulant with immunotropic activity, which can find application in biology and medicine.

 Known for their amino acid composition are analogs of tafcin, having a central activating or inhibitory effect, but lacking immunotropic activity.

 Known tetrapeptide Thr-Lys-Pro-Arg, tuftsin fragment of immunoglobulin, which has an immunotropic effect and has a short-term effect on the central nervous system (psychotropic effect). All of these compounds: tuftsin and its analogues have a short-term effect (15-20 minutes).

 The purpose of the invention is the search for new chemical compounds of peptide nature, which are long-acting psychostimulants and at the same time exhibiting the properties of an immunostimulant.

The goal is achieved by the described heptapeptide of the formula:
Thr-Lys-Pro-Arg-Pro-Gly-Pro, exhibiting the properties of a long-acting psychostimulant with immunotropic activity.

 The described compound is synthesized using classical methods of peptide chemistry by stepwise extension of the peptide chain, starting at the C-terminus of the peptide molecule. The synthesis is carried out using N, N'-dicyclohexylcarbodiimide with the addition of 1-hydroxybenzotriazole, as well as the method of pentafluorophenyl ethers, followed by cleavage of the protective groups by transfer hydrogenation with 1,4-cyclohexadiene in the presence of palladium black and hydrogen chloride in dioxane. The resulting peptide in the form of a hydrochloride salt is passed through a chromatographic column with Amberlist resin (AcO form) to exchange hydrogen chloride for acetate.

Accepted abbreviations:
Boc is a tert-butyloxycarbonyl group,
Bzl is a benzyl group,
BT - 1-hydroxybenzotriazole,
DCCH - dicyclohexylcarbodiimide,
DCM - dicyclohexylurea,
THF - tetrahydrofuran,
PFP - pentafluorophenyl ether,
TEA - triethylamine,
CHD - 1,4-cyclohexadiene,
EA - ethyl acetate,
TFA - trifluoroacetic acid,
DMF - dimethylformamide,
Z is carbobenzoxy.

Control over the synthesis of peptides is carried out on plates of the brand "Silufol" when spraying with a solution of ninhydrin in solvent systems:
1-butanolacetic acid - water 5: 1: 2;
chloroform-methanol 14: 1;
n-butanol - acetic acid - pyridine-water 30: 6: 20: 24;
chloroform - methanol-ammonia 6: 4: 1;
isobutanol - acetone - 12% ammonia;
isopropanol - formic acid - water 20: 1: 5.

 All solvents are absolute correspondingly. Melting points do not adjust. The target product after cleavage of the protective groups is subjected to acid cleavage by boiling in 6N. HCl for 48 h and then examined in solvent systems 5 and 6 by two-dimensional chromatography (fingerprint). In the hydrolysis product, all amino acids that make up the peptide are found.

 For purification and determination of heptapeptide homogeneity, high performance liquid chromatography (HPLC) was used on equipment of Du Pont (USA), a chromatograph, model 850, a Zorbax ODS column, 250 mm long and 4.6 in diameter; 9.4 and 21.2 mm, spectrophotometric UV detector (190-350 mm).

 The spectrum of circular dichroism is obtained on a Carry-60 spectropolarimeter with the prefix CD 001 while purging the optical part of the device with nitrogen.

 Optical rotation is measured on a Perkin Elmer digital polarimeter.

 The spectral characteristics of the final protected peptide and the target product are obtained on a Unicam SP 800 spectrophotometer within the wavelength range of 250-350 nm.

 PRI me R 1. Obtaining heptapeptide.

 1. N-tert-butyloxycarbonylpropylglycylproline benzyl ester.

0.930 g (4.3 mole) Boc-pro was dissolved in 25 mL of THF was added 0.580 g (4.3 mmol) TDB, cooled ^to 0 ° C and added 0.970 g DTSGK in 10 ml THF.

After stirring for 20 minutes while cooling, a solution of 1.68 g (4.3 mmol) of TFA glycyl-proline benzyl ester in 5 ml of THF and 0.6 ml (4.3 mmol) of TEA is added. The reaction mixture was stirred for 1 h at ⁰ C and overnight at room temperature, then the DCU filtered off and the solution evaporated in vacuo; the residue is dissolved in 250 ml of EA. The ethyl acetate solution was washed with 0.1 n. HCl solution, water, 5% NaHCO ₃ solution, H ₂ O, and then dried over MgSO ₄ and evaporated in vacuo. Dissolve the remaining oil in ether and reprecipitate with hexane. The viscous oil is triturated with ether and, after standing in the refrigerator overnight, the precipitate is filtered off and dried in vacuo.

Yield 1.350 g (68%). Mp 120-122 ^{° C;} R _f = 0.65 (1); 0.74 (2).

) аргинил-пролил-глицил-пролина.2. Tert-butyloxycarbonyl benzyl ester (NO ₂

) arginyl-prolyl-glycyl-proline.

A) 0.966 g of Boc Pro Gly Pro Bzl suspended in 6 mL CH ₂ Cl ₂ was added 6 mL of trifluoroacetic acid (TFA) and incubated for 30 min at room temperature, then evaporated in vacuo at 25-30 ^{° C,} repeatedly evaporated from abs. methanol with abs. ether, then fill in abs. ether and the separated precipitate after decantation of the ether is dried in vacuum.

Yield 0.95 g (2 mmol); R _f = 0.01 (1); 0.085 (2).

) Atg 0,638 г (2 ммоль) растворяют в 10 мл ТГФ и 2 мл ДМФ, добавляют 0,27 г (2 ммоль) БТ, охлаждают до 0^оС, и добавляют 0,46 г (2,2 ммоль) ДЦГК. После перемешивания в течение 40 мин при охлаждении, добавляют раствор 0,95 г (2 ммоль) ТФА Pro-Gly-Pro Bzl в 10 мл ТГФ и 0,28 мл (2 ммоль) ТЭА. Перемешивают реакционную смесь в течение трех суток. Отфильтровывают осадок ДЦМ, упаривают раствор в вакууме, а к остатку приливают 150 мл гексана, при этом отделяется целевой продукт в виде масла, которое растворяют в 200 мл ЭА и обрабатывают как на стадии 1.B) Boc (NO ₂

) Atg 0,638 g (2 mmol) is dissolved in 10 ml of THF and 2 mL of DMF was added 0.27 g (2 mmol) of WT, cooled ^to 0 ° C and add 0.46 g (2.2 mmol) DTSGK. After stirring for 40 minutes while cooling, a solution of 0.95 g (2 mmol) of TFA Pro-Gly-Pro Bzl in 10 ml of THF and 0.28 ml (2 mmol) of TEA is added. Stir the reaction mixture for three days. The DCM precipitate is filtered off, the solution is evaporated in vacuo, and 150 ml of hexane are added to the residue, while the target product is separated in the form of an oil, which is dissolved in 200 ml of EA and treated as in stage 1.

Yield 0.902 g (85%). Mp 108-110 ^about C;
[α] _D ²² - 77.8 ^° (C = 0.5, AcOH);
R _f 0.44 (1); 0.80 (3).

) пролил-глицил-пролина.3. Tert-butyloxycarbonyl-prolyl-arginyl benzyl ester
(NO ₂

) prolyl-glycyl-proline.

A) 0.982 g (1.63 mmol) was suspended in 4 ml of CH ₂ Cl ₂ was added 4 mL TFA and allowed to stand 45 min at room temperature, evaporated in vacuo at 25 ^C. The residue was evaporated several times with abs. EtOH, then with abs. ether, then adding abs. ether precipitate, which is washed with decantation of abs. ether and then dried in vacuo.

 The output is quantitative.

R _f 0.16 (1); 0,0 (2); 0.27 (3).

B. Boc-pro 0,352 g (1.63 mmol) was dissolved in 5 ml of THF was added 0.22 g (1.63 mmol) TDB, cooled ^to 0 ° C and 0.37 g DTSGK in 10 ml THF. After 40 minutes, a solution of TFA Arg (NO ₂ ^ω ) - Pro-Gly-ProOBzl (1.63 mmol) in 10 ml THF and 0.230 ml (1.63 mmol) TEA was added to the reaction mixture. Stirring 2 hours at 0 ^C and two days at room temperature, then the DCU precipitate is filtered, evaporated in vacuo, dissolved in 100 ml of EA and treated similarly to step 2 B.

Yield 840 mg (67.8%). Mp 147-148 ^about C;
R _f - 0.42 (1); 0.31 (2); 0.72 (3)
[α] _D ²² = -66.0 ^° [C = 0.5; AcOH].

4. Pentafluorophenyl ether of tert-butyloxycarbonyl-ε-carbobenzoxylisine (Boc-Lys (Z) ^ε PFP).

To Boc-Lys (Z) ^ε 0.76 g (2 mmol) in 5 ml abs. EA was added 0.50 g (2.7 mmol) of pentafluorophenol, cooled ^to 0 ° C and add 0.50 g (2.4 mmol) DTSKG in 10 ml THF. The reaction mixture was stirred for 2 hours at ⁰ ° C, DCU was filtered off, the solution was evaporated in vacuo, added 5 ml of abs. ether and 25 ml of hexane, a precipitate gradually precipitates upon cooling, which is filtered off and dried in vacuo.

Yield 0.454 g (87%). Mp 117-120 ^about C;
R _f 0.84 (1); 0.91 (2).

)-пролил-глицил-пролина.5. Tert-butyloxycarbonyl-lysyl- [ε-carbobenzoxy] -propyl-arginyl benzyl ester (NO ₂

) -prolyl-glycyl-proline.

)-Pro-Gly-Pro OBzl.A. Boc-Pro-Arg (NO ₂

) -Pro-Gly-Pro OBzl.

0.84 g (1.1 mmol) in 3 ml of CH ₂ Cl ₂ and 3 ml of TFA are treated as in stage 3, A.

Yield 0.813 g (1.07 mmol, 97%); R _f 0.095 (1).

)-Pro-Gly-Pro OBzl.5. CTFA - Pro-Arg (NO ₂

) -Pro-Gly-Pro OBzl.

0.813 g (1.07 mmol) dissolved in 10 ml of THF and cooled ^to 0 ° C, was added with stirring 0.15 ml of TEA and then for 2 hours with four portions of ^{Boc-Lys (Z) ε PFP} 0,585 mg (1 07 mmol). After stirring for three days at room temperature, the reaction mixture was evaporated in vacuo, the residue was dissolved in 60 ml of EA and treated as in step 1.

Yield 0.60 g (58%). Mp 130-132 ^about S.

R _f 0.38 (1); 0.75 (3).

[α] _D ²² - 86.2 ^o (c 0.5; Ac OH).

)-пролил-глицил-пролина.6. Benzyl ester tert-butyloxycarbonyl-threonyl-lysyl- (ε-carbobenzoxy) -prolyl-arginyl- (NO ₂

) -prolyl-glycyl-proline.

)-Pro-Arg (NO₂

)-Pro-Gly-Pro OBzl.A) Boc Lys- (Z

) -Pro-Arg (NO ₂

) -Pro-Gly-Pro OBzl.

0.69 g (0.69 mmol) in 2 ml of CH ₂ Cl ₂ and 2 ml of TFA are treated as in step 3 A.

 Yield: quantitative.

R _f 0.12 (1); 0.45 (3).

)-Pro-Arg(NO₂

) -Pro-Gly-Pro OBzl.B) To TFA Lys (Z

) -Pro-Arg (NO ₂

) -Pro-Gly-Pro OBzl.

(0.69 mmol) in 30 mL of THF while cooling ^to 0 ° C was added 0.097 ml of TEA and 0.266 g of Boc Thr PFP, prepared as in step 4. Dalee the desired product was synthesized as in step 5, B, was added during the reaction to TEF pH 6-7.

Yield 0.616 g (80%). Mp 155-156 ^about C;
R _f 0.39 (1); 0.145 (2); 0.80 (3); 0.96 (4);
[α] _D ²² - 79.2 ^o (c = 0.5; Ac OH).

 7. Tert-butyloxycarbonyl-threonyl-lysyl-prolyl-arginyl-prolyl-glycyl-proline.

) Pro-Gly-Pro OBzl кипятят в 3 мл абс. EtOH и 4 мл 1,4-циклогексадиена в течение 4-5 ч в присутствии 55 мг Pd-черни (контроль - хроматографирование на пластинках "Силуфол", далее спектрофотометрический анализ выделенного вещества на отщепление NO₂

группы как наиболее трудно отщепляемой каталитическим восстановлением), катализатор отфильтровывают, многократно промывают этанолом. Объединенный фильтрат упаривают досуха, а остаток дважды переосаждают из MeOH-эфиром.0.170 g (0.152 mmol) Boc-Thr-Lys (Z ^ε ) -Pro-Arg (NO ₂

) Pro-Gly-Pro OBzl is boiled in 3 ml abs. EtOH and 4 ml of 1,4-cyclohexadiene for 4-5 hours in the presence of 55 mg of Pd-black (control - chromatography on Silufol plates, then spectrophotometric analysis of the isolated substance for NO ₂ cleavage

group as the most difficult to split off by catalytic reduction), the catalyst is filtered off, washed repeatedly with ethanol. The combined filtrate was evaporated to dryness, and the residue was reprecipitated twice from MeOH-ether.

Yield 106.4 mg (94% R _f = 0.125 (1); 0.37 (3), 0.57 (4); [α] _D ²² -75.6 ^o (c = 0.25; Ac OH) .

 8. Threonyl-lysyl-prolyl-arginyl-prolyl-glycyl-proline.

 90.4 mg of Boc-Thr-Lys-Pro-Arg-Pro-Gly-Pro is suspended in 1.5 ml of 2N. HCl (dioxane and incubated at room temperature for 45 minutes. Then dry ether is added, evaporated in vacuo, dry ether is added again and the precipitate is washed several times with decantation with dry ether. Precipitated from MeOH-ether.

 The resulting precipitate (86 mg) was dissolved in 1.5 ml of 30% EtOH and applied to a column (V = 1 ml) of Amberlist A-21 (AcO form) to exchange the hydrochloride salt for acetate. Elute peptide ≈ 30-40 ml. 30% EtOH, evaporated to dryness in vacuo and precipitated from methanol - ether.

Yield 65 mg (75%);
R _f 0.01 (1); 0.22 (4); 0.10 (3);
[α] _D ²² = -62.8 ^° (c = 0.25; AcOH).

 Amino acid analysis: Thr 0.9 (1); Lys 0.98 (1); Pro 2.78 (3); Arg 1.0 (1); Gly 1.1 (1).

Further purification of the obtained peptide is performed by high performance liquid chromatography (HPLC). Gradient solvent B 0-60% for 60 min [solvent A: 0.01 M CH ₃ COONH ₄ , pH 4.0, solvent B: 0.01 M CH ₃ COONH ₄ (A) in MeOH], 50 ^° C, a flow rate of 4 or 10 ml / min for columns with a diameter of 9.4 and 21.2 mm, respectively.

Analytical chromatography solvent A: 0.05 M NH ₄ H ₂ PO ₄ (pH 4.0); solvent B: MeOH, solvent gradient 0-50% B over 50 min, 50 ° ^C, flow rate 1 mL / min, column diameter 4.6 mm.

.The spectrum of circular dichroism was obtained on a Carry-60 spectropolarimeter. The peptide concentration of 0.1 mg / ml H ₂ About, the sleepers - 0.1 ^about , the copy speed of 5 nm / min, the resolution of the gap 15

.

 Biological tests of the described heptapeptide were carried out.

 The studied peptides were administered intraperitoneally in an aqueous solution to male nonlinear white rats (weighing ≈ 250 g) and mice (weighing ≈ 20 g) 7, 14, and 30 min before the experiment. To the control animals, distilled water in the same volume was injected in the same way.

 1. Radiation of the action of peptides on the implementation of behavior - ridding rats of a stressful situation.

 Rats previously divided into groups according to the nature of their emotional response (1-non-emotional, 2-emotional) are placed in a cylinder with water. Avoidance can be done by diving under the walls of the cylinder. The following indicators are recorded: latent period (bc), implementation of disposal behavior, number of errors (incomplete diving attempts), the nature of the emotional response, the total duration of the stimulating effect.

 Heptapeptide in a wide range of doses (70-1200 μg / kg) reduced, and in doses of 900-1000 μg / kg completely eliminated errors in the implementation of the behavior of the disposal of rats of group 2. The latent period of implementation of avoidance was reduced by 30 or more times. A dose-dependent effect was observed. The optimizing effect on the behavior of ridding rats of the 1st group (non-emotional) developed against the background of large doses (1000-1200 μg / kg). The effect was maintained within 60-90 minutes. Tufcin tetrapeptide had a similar, but short-term (15 min) effect, without a dose-dependent effect and without regard to the type of emotional response.

 2. The study of the action of peptides on the behavior of "despair" (refusal of activity, immobilization) of forced swimming mice.

 Mice are placed in a closed cylinder with water without the possibility of avoidance. Estimate the duration of maintaining a fixed position on the water (immobilization) for different periods of time 6, 12, 30 minutes).

 Heptapeptide sharply reduced the period of immobilization. The effect was maintained at all recorded time intervals. Tuftsin was effective only in the first 4-6 minutes and in a narrow dose range.

 The results are shown in table. 1.

 The data indicate that the psychostimulating effect of the heptapeptide is 3 times greater in intensity than tufcin, 1.5 times in the range of effective doses, and 3-4 times in duration of action.

 3. The study of the immunotropic activity of peptides.

 Phagocytic activity was studied in a single-layer culture of muscle peritonial macrophages using Salm phagocytase as an object. thyphimurium, as well as in blood smears of patients with reduced antibody production.

 The heptapeptide was shown to increase the phagocytic activity of macrophages by 19%, surpassing tuftsin by 9%. The rate of completion of phagocytosis (PZF) increases 1.5 times stronger than against the background of tufcin. The number of phagocytically active cells increases by 50% (table. 2).

определена токсичность описываемого гептапептида.Phagocytosis completion rate:
IF - Σ

The toxicity of the described heptapeptide was determined.

 The experiment was performed on 30 experimental animals (mice) weighing 20 g with intraperitoneal administration of heptapeptide in doses of 0.5; 5; 50 mg / kg, i.e. 10, 100, 1000 times higher than effective. Observations are carried out 60 mi after administration on the same day and daily for the next 5 days. In no case was a toxic effect detected by the lateral position and the myrolaxing effect. At autopsy, no negative effect on the structure of internal organs was found. It was found that the described heptapeptide does not have a toxic effect on animals at a dose exceeding the effective (0.05 mg / kg) 1000 times.

 The studies conducted allow us to conclude that the described compound is 3-4 times superior to tuftsin in duration of psychostimulating action, in 3 times in intensity, by 1.5 times in effective dose range, and by 1.5 times in terms of phagocytosis completeness. Moreover, the described compound has low toxicity. All this suggests the possibility of its use in biology and medicine as a long-acting psychostimulator with immunotropic activity.

Claims (1)

 HEPTAPEPTIDE Possessing the Properties of a Psycho-stimulator of Prolonged Action with Immunotropic Activity. Heptapeptide Formula
Thr - hys - Pro - Arg - Pro - GLy - Pro, which has the properties of a long-acting psychostimulant with immunotropic activity.

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